Peptide Quantitation with Direct Detect® Spectrometer

Fast and Accurate Peptide Quantitation Using the Direct Detect® Spectrometer

Traditional peptide quantitation can be inaccurate.

Weight-based quantitation can overestimate amounts of lyophilized peptides. Given that peptide concentrations obtained using the Direct Detect® spectrometer can be verified by amino acid analysis, these data indicate that weight-based peptide quantitation may overestimate peptide concentrations in many samples. (click to enlarge)

Determining peptide concentration accurately and quickly has proven difficult for many researchers. Most commonly used methods for peptide quantitation rely on the weight of the lyophilized powder, absorbance of ultraviolet (UV) light or amino acid analysis. Establishing peptide concentration based on the weight of the lyophilized peptide is inaccurate in most cases, because the analyzed powder can contain a significant quantity (10-70%) of bound water, salts or counterions. Another peptide quantitation method relies on absorbance at 280 nm, and thus can only be used to estimate peptide concentration if tryptophan and tyrosine resides are present in the sequence. Therefore, peptides that do not contain amino acids that absorb light at 280 nm cannot be accurately quantified using this method.

Benefits of Using Direct Detect® Spectrometer for Peptide Quantitation

Two peptides quantified using the Direct Detect® spectrometer calibrated using BSA and two ACTH peptide standards. All three methods resulted in concentrations matching results of amino acid analysis (AAA). (click to enlarge)

The Direct Detect® spectrometer provides a universal, fast and accurate peptide quantitation method that does not require sample manipulation. IR spectroscopy exploits the fact that molecules absorb specific frequencies characteristic of their structure. To form a peptide, amino acids are covalently linked via amide (peptide) bonds. In order to determine protein and peptide concentration, the Direct Detect® spectrometer uses the intensity of the Amide I band, which is assigned to C=O stretching vibration of the peptide bond (about 80%) with a minor contribution from C-N stretching vibration (about 20%).